Methods for preparing transgenic animals are extremely important in the basic and applied researches. Up to present, scientists have developed many transgenic technologies to improve the targeting and integration efficiency in transgenic animals. However, in large animals, the transgenic integration efficiency and expression rate are not satisfactory.
The current universal transgenic method in the world is microinjection, which has high costs and low success rate. For a transgenic animal or a mammary gland bioreactor, the success rate can only reach about 3%. Microinjection results in random integration of an exogenous gene in chromosomes, and the transgenic expression is greatly influenced by the surrounding host genome. The random integration of an exogenous gene may cause the following questions. 1. The probability for integrating a transgene into inactive chromosomal sequences is higher than that for integrating into an active chromosomal region and most of transgenes are low expressed or not expressed at all because of the influence from the integration site. 2. When the transgene integrates into a high-frequency recombination site, it may cause genetic instability. 3. Random integration of an exogenous gene can often lead to mutation or altered expression of endogenous genes, thus effecting normal development and health of transgenic animals. 4. For a transgenic animal with integration in multiple sites, the isolation of integration sites in its offspring may lead to property change of transgenic expression and difficulty in breeding. 5. A significantly high or low copy number of integration may easily cause low or no transgenic expression.
The above questions are key barriers in the studies of gene function, creation of animal disease models, development of commercially valuable models and creation of transgenic breeding new materials. Developing a method for positioning and integrating transgene may overcome negative effects of high costs, low efficiency caused by the traditional technology, and promote industrial development of transgenic livestock. However, there is no method for controlling target gene to efficiently integrate into a specific site of genome. Therefore, there is an urgent need in the art to develop an efficient method for positioning and integrating transgene.